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A multi-scale approach reveals that NF-κB cRel enforces a B-cell decision to divide.

Shokhirev MN, Almaden J, Davis-Turak J, Birnbaum HA, Russell TM, Vargas JA, Hoffmann A - Mol. Syst. Biol. (2015)

Bottom Line: B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically.Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells.We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Signaling Systems Laboratory, UCSD, La Jolla, CA, USA San Diego Center for Systems Biology, UCSD, La Jolla, CA, USA Bioinformatics and Systems Biology Graduate Program, UCSD, La Jolla, CA, USA.

No MeSH data available.


Related in: MedlinePlus

Molecular assays suggest that NF-κB enforces an upstream fate decisionA–L Naïve purified B cells were stimulated with 250 nM CpG for 24 h and analyzed using single-cell RNA sequencing (A top). Five small and five large B cells were captured in a microfluidics chip (B), and their transcriptomes were sequenced to reveal sets of genes typically upregulated in big cells (C, red) or small cells (C, green). Pathway analysis on genes upregulated in large B cells (D, top) and small cells (D, bottom) was performed. (E) Transcription factor motif enrichment analysis on the genes upregulated in large cells (E, top) and small cells (E, bottom) was performed and filtered to show only significantly upregulated (P < 0.05) and known NF-κB target genes or NF-κB itself. NF-κB cRel abundances of purified naïve B cells stimulated with 250 nM CpG for 24 h were obtained by quantifying average fluorescence in fixed B cells stained with anti-cRel antibody conjugated to fluorophore, or anti-BclXL antibody bound to a fluorescent secondary antibody (A bottom). The 0-h average fluorescence was used to determine significant upregulation of NF-κB cRel (F), growth regulator Myc (H), and anti-apoptotic regulator BclXL (J) at 24 h (P < 0.05). Immunoblot for p-S6 (arrow), a downstream target of mTORc1, with anti-tubulin control after 24 h CpG stimulation in WT and cRel-deficient B cells and gel quantification is shown (L). NF-κB cRel-deficient cells were used to approximate the technical noise (G) or dependence of Myc (I), BclXL (K), and mTORc1 (L) on NF-κB cRel. Quadrants in (F–K) indicate fraction of cells at 24 h compared to 0 h. Growth was manually defined as a cell area > 100 pixels to avoid cell selection bias in images.
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fig04: Molecular assays suggest that NF-κB enforces an upstream fate decisionA–L Naïve purified B cells were stimulated with 250 nM CpG for 24 h and analyzed using single-cell RNA sequencing (A top). Five small and five large B cells were captured in a microfluidics chip (B), and their transcriptomes were sequenced to reveal sets of genes typically upregulated in big cells (C, red) or small cells (C, green). Pathway analysis on genes upregulated in large B cells (D, top) and small cells (D, bottom) was performed. (E) Transcription factor motif enrichment analysis on the genes upregulated in large cells (E, top) and small cells (E, bottom) was performed and filtered to show only significantly upregulated (P < 0.05) and known NF-κB target genes or NF-κB itself. NF-κB cRel abundances of purified naïve B cells stimulated with 250 nM CpG for 24 h were obtained by quantifying average fluorescence in fixed B cells stained with anti-cRel antibody conjugated to fluorophore, or anti-BclXL antibody bound to a fluorescent secondary antibody (A bottom). The 0-h average fluorescence was used to determine significant upregulation of NF-κB cRel (F), growth regulator Myc (H), and anti-apoptotic regulator BclXL (J) at 24 h (P < 0.05). Immunoblot for p-S6 (arrow), a downstream target of mTORc1, with anti-tubulin control after 24 h CpG stimulation in WT and cRel-deficient B cells and gel quantification is shown (L). NF-κB cRel-deficient cells were used to approximate the technical noise (G) or dependence of Myc (I), BclXL (K), and mTORc1 (L) on NF-κB cRel. Quadrants in (F–K) indicate fraction of cells at 24 h compared to 0 h. Growth was manually defined as a cell area > 100 pixels to avoid cell selection bias in images.

Mentions: To characterize the molecular connections that underlie fate decision processes, we turned to single-cell molecular assays (Fig4A). Following CpG stimulation of B cells for 24 h, we sequenced the transcriptomes of five large and five small cells using a single-cell autoprep system, which allowed us to image and measure the size of individual B cells trapped in a microfluidic chip (Fig4B). After normalizing transcript counts to RNA spike-in controls, we identified 369 upregulated and 121 downregulated genes in large versus small cells (Fig4C). Using pathway enrichment tools, we identified pathways that were significantly upregulated in large cells, including metabolism, the control of apoptosis and the G1-to-S transition, and NF-κB, a known key regulator of B-cell expansion (Fig4D). Further, we performed a transcription factor enrichment analysis on the upregulated and downregulated gene sets and found that binding motifs of nine transcription factors that are known NF-κB target genes, as well as NF-κB itself, were enriched among the genes upregulated in big cells (Fig4E), while p53 was the only known NF-κB target gene transcription factor enriched in the set of genes downregulated in big cells.


A multi-scale approach reveals that NF-κB cRel enforces a B-cell decision to divide.

Shokhirev MN, Almaden J, Davis-Turak J, Birnbaum HA, Russell TM, Vargas JA, Hoffmann A - Mol. Syst. Biol. (2015)

Molecular assays suggest that NF-κB enforces an upstream fate decisionA–L Naïve purified B cells were stimulated with 250 nM CpG for 24 h and analyzed using single-cell RNA sequencing (A top). Five small and five large B cells were captured in a microfluidics chip (B), and their transcriptomes were sequenced to reveal sets of genes typically upregulated in big cells (C, red) or small cells (C, green). Pathway analysis on genes upregulated in large B cells (D, top) and small cells (D, bottom) was performed. (E) Transcription factor motif enrichment analysis on the genes upregulated in large cells (E, top) and small cells (E, bottom) was performed and filtered to show only significantly upregulated (P < 0.05) and known NF-κB target genes or NF-κB itself. NF-κB cRel abundances of purified naïve B cells stimulated with 250 nM CpG for 24 h were obtained by quantifying average fluorescence in fixed B cells stained with anti-cRel antibody conjugated to fluorophore, or anti-BclXL antibody bound to a fluorescent secondary antibody (A bottom). The 0-h average fluorescence was used to determine significant upregulation of NF-κB cRel (F), growth regulator Myc (H), and anti-apoptotic regulator BclXL (J) at 24 h (P < 0.05). Immunoblot for p-S6 (arrow), a downstream target of mTORc1, with anti-tubulin control after 24 h CpG stimulation in WT and cRel-deficient B cells and gel quantification is shown (L). NF-κB cRel-deficient cells were used to approximate the technical noise (G) or dependence of Myc (I), BclXL (K), and mTORc1 (L) on NF-κB cRel. Quadrants in (F–K) indicate fraction of cells at 24 h compared to 0 h. Growth was manually defined as a cell area > 100 pixels to avoid cell selection bias in images.
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Related In: Results  -  Collection

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fig04: Molecular assays suggest that NF-κB enforces an upstream fate decisionA–L Naïve purified B cells were stimulated with 250 nM CpG for 24 h and analyzed using single-cell RNA sequencing (A top). Five small and five large B cells were captured in a microfluidics chip (B), and their transcriptomes were sequenced to reveal sets of genes typically upregulated in big cells (C, red) or small cells (C, green). Pathway analysis on genes upregulated in large B cells (D, top) and small cells (D, bottom) was performed. (E) Transcription factor motif enrichment analysis on the genes upregulated in large cells (E, top) and small cells (E, bottom) was performed and filtered to show only significantly upregulated (P < 0.05) and known NF-κB target genes or NF-κB itself. NF-κB cRel abundances of purified naïve B cells stimulated with 250 nM CpG for 24 h were obtained by quantifying average fluorescence in fixed B cells stained with anti-cRel antibody conjugated to fluorophore, or anti-BclXL antibody bound to a fluorescent secondary antibody (A bottom). The 0-h average fluorescence was used to determine significant upregulation of NF-κB cRel (F), growth regulator Myc (H), and anti-apoptotic regulator BclXL (J) at 24 h (P < 0.05). Immunoblot for p-S6 (arrow), a downstream target of mTORc1, with anti-tubulin control after 24 h CpG stimulation in WT and cRel-deficient B cells and gel quantification is shown (L). NF-κB cRel-deficient cells were used to approximate the technical noise (G) or dependence of Myc (I), BclXL (K), and mTORc1 (L) on NF-κB cRel. Quadrants in (F–K) indicate fraction of cells at 24 h compared to 0 h. Growth was manually defined as a cell area > 100 pixels to avoid cell selection bias in images.
Mentions: To characterize the molecular connections that underlie fate decision processes, we turned to single-cell molecular assays (Fig4A). Following CpG stimulation of B cells for 24 h, we sequenced the transcriptomes of five large and five small cells using a single-cell autoprep system, which allowed us to image and measure the size of individual B cells trapped in a microfluidic chip (Fig4B). After normalizing transcript counts to RNA spike-in controls, we identified 369 upregulated and 121 downregulated genes in large versus small cells (Fig4C). Using pathway enrichment tools, we identified pathways that were significantly upregulated in large cells, including metabolism, the control of apoptosis and the G1-to-S transition, and NF-κB, a known key regulator of B-cell expansion (Fig4D). Further, we performed a transcription factor enrichment analysis on the upregulated and downregulated gene sets and found that binding motifs of nine transcription factors that are known NF-κB target genes, as well as NF-κB itself, were enriched among the genes upregulated in big cells (Fig4E), while p53 was the only known NF-κB target gene transcription factor enriched in the set of genes downregulated in big cells.

Bottom Line: B-lymphocyte population dynamics, which are predictive of immune response and vaccine effectiveness, are determined by individual cells undergoing division or death seemingly stochastically.Combining modeling and experimentation, we found that NF-κB cRel enforces the execution of a cellular decision between mutually exclusive fates by promoting survival in growing cells.We show that a multi-scale modeling approach allows for the prediction of dynamic organ-level physiology in terms of intra-cellular molecular networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Signaling Systems Laboratory, UCSD, La Jolla, CA, USA San Diego Center for Systems Biology, UCSD, La Jolla, CA, USA Bioinformatics and Systems Biology Graduate Program, UCSD, La Jolla, CA, USA.

No MeSH data available.


Related in: MedlinePlus